1. Field of the Invention
The present invention relates to optical detection of macromolecular components separated in a capillary column, and more particularly to the alignment of the capillary column with the detection optics.
2. Description of Related Art
Macromolecules of a biological sample can be separated in a capillary column by means of several techniques. One of the techniques is capillary electrophoresis in which a sample is separated in a capillary channel by application of a high electric potential along the capillary channel. The sample separates into zones of macromolecule species as a result of different mobilities within the separation medium that are inherent to the macromolecules, depending on their electric charges, sizes, and shapes.
The separated zones can be detected by several techniques. One of many optical detection techniques is laser induced fluorescence detection. Reference is made to U.S. Pat. No. 4,675,300 to Zare et al for a detailed description of the detection technique. In general light from a laser source is passed through a section of a capillary separation channel to cause the separated sample components which were tagged with fluorescent material prior to, during or just after separation to fluoresce. The fluorescence may be detected with high sensitivity. High sensitivity and maximum resolution are achieved by concentrating the laser beam into a narrow spot which interrogates a short section of the capillary separation channel defining a small detection volume. A typical size for the spot is on the order of 50 microns or less for a separation channel having a diameter of the same order or larger.
Like any other optical detection scheme, maximum sensitivity is achieved for laser induced fluorescence detection only when the entire configuration of the laser beam, capillary separation channel and optical elements are properly aligned. A critical component of proper optical alignment is the positioning of the capillary detection volume with respect to the laser beam. Because typical detection volumes and capillary dimensions are extremely small, misalignment of the capillary channel and the laser beam by only a few microns may seriously degrade detector performance.
It is difficult to correctly position the capillary in the laser beam to achieve perfect alignment, and to maintain this position once it is achieved. Factors compounding this difficulty include the requirement that capillaries be readily replaceable, the fact that the capillary may flex under application of high electric fields, and the fact that alignment may drift as a result of thermal fluctuations. The exact cause of the capillary motion is uncertain. One suspects that the charge distribution in the capillary channel accompanied by time-dependent changes in the electric field at the inside walls of the capillary column may be part of the cause.
One current approach to aligning an open-tube capillary (filled with electrolyte solution as opposed to packed capillary which are filled with gel electrolyte or the like materials) prior to an electrophoresis run is to pump into the capillary a fluorescent solution and adjust the alignment while monitoring the fluorescence with either a photomultiplier tube or a photodiode. Alignment is considered achieved when the fluorescence intensity is at a maximum.
The above-described means of alignment is applicable only to open tube capillaries, i.e. filled only with liquid electrolyte, not gel, since fluorescent solution cannot be effectively pumped into a gel-filled column before alignment and later completely removed from the gel after alignment. This means also is undesirable in an open tube because it is often difficult or impossible to completely remove the fluorescent material from the capillary inner walls afterwards. Furthermore, there is no provision for performing dynamic alignment during an electrophoretic process. Since the capillary column may misalign laterally under thermal fluctuation or the high applied electric potential, there is no way of knowing when the alignment has drifted away from optimum during an electrophoresis operation.